AU643502B2

AU643502B2 - Expression of a multigene RNA with self-splicing activity

Info

Publication number: AU643502B2
Application number: AU71870/91A
Authority: AU
Inventors: Hubert Mullner; Rudolf Schneider; Eugen Uhlmann
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1990-02-01
Filing date: 1991-01-25
Publication date: 1993-11-18
Anticipated expiration: 2011-01-25
Also published as: WO1991011511A1; DE59103546D1; HU9202483D0; EP0513133B1; HUT62324A; EP0513133A1; KR100240356B1; CA2075180A1; ES2066422T3; DE4002885A1; NZ236945A; OA09663A; AU7187091A; JPH05504064A; KR927003799A

Description

HOECHST AKTIENGESELLSCHAFT Dr.SI/gm HOE 90/F 034 Description Expression of a multigene RNA having self-splicing activity RNA molecules can, under suitable conditions, catalyze reactions on other RNA molecules or autocatalytically cleave off fragments from their own molecules without the participation of proteins. For example, an intron having 413 nucleotides is deleted autocatalytically at the 3' end of the 23s rRNA of Tetrahymena thermophila and transformed into a circular form. This takes place by means of a number of phosphoester transfer reactions with guanosine cofactors participating (Cech, Nature 578-583 (1983)). Depending on the RNA substrate or the reaction conditions chosen, the intron can function as specific ribonuclease, terminal transferase, phosphotransferase or acid phosphatase. In this connection, an RNA molecule can carry out a several conversions without being changed itself and has the characteristics of an enzyme in this respect. For this reason, the term ribozyme has been introduced for RNA molecules having these properties.

It was possible to show similar reactions without participation of proteins also for some viroid RNAs and satellite RNAs. For example for avocado sunblotch viroid (ASBV) (Hutchins, C.J. et al. Nucleic Acids Res. 14, 3627-3640 (1986)), satellite RNA of tobacco ringspot virus (sTobRV) (Prody, G.A. et al., Science 231, 1577-1580 (1986)) and satellite RNA of luzerne transient streak virus (sLTSV) (Forster A.C. et al., Cell 49, 211- 220 (1987)), self-processing seems to be a reaction essential for multiplication. During the replication of these RNAs circular forms are presumably formed which, as templates, lead to the synthesis of RNAs which are overlong. These transcripts are cut to the right genome 2 length by the self-catalyzed endonucleolytic reactions.

The structures of the RNAs, which these presumably take on for the reaction, have been described as hammerheads (Forster A.C. et al., Cell 49, 211-220 (1987)); Haseloff, J. et al., Nature 334, 585-591 (1988)).

The cleavage sites for these RNA enzymes are specific and must have certain structural prerequisites in order to allow processing.

Surprisingly, it has now been found that host cells of any desired organisms can be transformed using vectors which contain DNA coding for ribozyme RNA linked to functional genes, so that said RNA is expressed and subsequently spliced.

The invention thus relates to: 1. A hybrid gene comprising one or more copies of a gene sequence coding for ribozyme RNA and of one or various functional genes, the gene sequences being linked via a spacer which, on the RNA level, represents a substrate for the ribozyme.

2. Host cells containing the gene characterized in 1.

In the following, the invention is described in detail, in particular in its preferred embodiments. Furthermore, the invention is defined by the contents of the claims.

A functional gene is a DNA section in the genome, which codes for a polypeptide. The polypeptide can, on its own, be a functional protein, or function as sub-unit of an enzyme complex.

'4~j The gene according to the invention is constructed in O( such a way that, starting from a promoter, several genes, for example entire synthetic pathways for the production 3 of e.g. amino acids, such as glycine, nucleotides, secondary metabolites such as antibiotics, cofactors of enzymes, hormones, such as thyroid hormone, can be expressed in plants or microorganisms. The intention of this is, on the one hand, to produce foreign substances in appropriate plants or microorganisms and, on the other hand, to increase the yield in plants or microorganisms which naturally synthesize these substances. Genes coding for appropriate polypeptides can be employed according to the invention by using control sequences.

It is furthermore possible to employ several copies of a gene for a particular functional protein, e.g. in order to increase the yield of a protein such as insulin or transaminase.

Furthermore, the expression of one or more selection markers using the system according to the invention is possible. For this purpose, for example, genes for the following proteins can be employed: 3-lactamase, -galactosidase, phosphinotricine acetyltransferase, chloramphenicol acetyltransferase or thymidine kinase.

The acetyltransferase gene for the herbicide phosphinotricine and the Cat gene from the transposon Tn9 for the chloramphenicol acetyltransferase are preferably used.

The acetyltransferase gene from Streptomyces viridochromogenes (Wohleben, W. et al., Gene 70, 25-37 (1988)), can be assembled from synthetically prepared oligonucleotides, there also being the possibility of modifying it for the expression in plants. The gene gives resistance to phosphinotricine to transgenic plants which express the product constitutively. The Cat gene effects resistance to chloramphenicol. Like the acetyltransferase gene, the Cat gene acetylates its product. The Cat gene pLUA, is derived from Tn9 (Alton, N.K. Pt al., Nature 15 282, 864-866, 1979) but is also commercially available.

4 The genes are linked by ribozyme substrate sequences, so-called spacers, and are released by a ribozyme structure domain of the same molecule. In this way, at least 2 and up to about 25 or more gene sequences can be expressed in one organism. The release can also take place via a separately expressed ribozyme molecule.

The appropriately transcribed RNA is essentially composed so that the ribozymes are preferably at the 3' or 5' end of the RNA molecule. A sequence of 40-50 nucleotides, which is entirely or in a partial region comprising at least 10, preferably 15-35, nucleotides complementary to the sequence of the ribozyme, is inserted as spacer. The ribozyme sequence of the RNA can, in this way, associate itself with the spacer and cut the latter immediately downstream of a defined sequence. A GUC triplet is preferably used as ribozyme cleavage site. The number of linked genes can, in each case, be multiplied by introducing further spacers, it being possible for the sequenc of the latter to be the same or different from the first spacer introduced. If it is different, a further ribozyme structure domain matching this sequence is created in the same RNA molecule.

The spacer and ribozyme sequences necessary for this RNA can be prepared synthetically. The linkage to the genes is carried out by means of suitable linkers which have been synthesized on.

Spacers and ribozyme are synthesized to be analogous to ribozyme structures in nature (Uhlenbeck, O.C., Nature 328, 596-600, (1987). In this connection, these sequences can mimic naturally occurring ribozymes (Forster A.C. et al., Cell 49, 211-220, (1987)) or be constructed in such a way that the essential structures of the ribozyme axe present, but that other sequences are S chosen for nonessential parts. In the basic construction, the procedure by Haseloff, J. et al., Nature 334, 585- 591, 1988 can essentially be carried out. A GUC sequence 5 around which sequences which are complementary to a described ribozyme sequence are located in the direction of 5' and 3' is incorporated into the spacer part of the

RNA.

Spacer and ribozyme on the RNA level can diagrammatice .ly be described as follows: NNNNNNNNNNNNNN-3 Substrate RNA 3-KKKKKKKKCA

AC

A U CG G VV V Ribozyme

VV

\L\

where N are nucleotides of the substrate RNA, A, C, G or U K are nucleotides complementary to N in the ribozyme V are variable nucleotides A, C, G or U in the ribozyme and VL are variable nucleotides A, C, G or U in the loop of the ribozyme.

The number of nucleotides of V, can be 0-550.

The gene according to the invention is cloned int, an intermediary vector having a plant promoter. Vectors of this type are for example the plasmids pNCN (Fromm M. et al., PNAS 82, 5824-5826 (1985) or pNOS (An G. et al.

EMBO J. 4, 277-276 (1985), or preferably pDH51 (Pietrzak, M. et al. NAR 14, 5857-5861, (1986).

After subsequent transformation of E. coli, such as e.g.

SE. coli MC 1061, DH1, DK1, GM48 or XL-1, positive clones 6 are identified by methods known per se (Maniatis et al., Lab. Manual), such as plasmid minipreparation and cleavage using an appropriate restriction enzyme. These positive clones are then subcloned in a binary plant vector. pGV3850 (Zambrysky, P. et al., EMBO J. 2, 2143- 2150 (1983)) or pOCA18 (Olszewski, NAR 16, 10765- 10782, (1988)) can be employed as plant vectors. Advantageously pOCA18 is used.

The binary plant vectors obtained which contain in the T-DNA a plant promoter with the attached DNA fragment which is constructed as specified above are used to transform plants. This can be carried out by techniques such as electroporation or microinject.on.

Preferably, the cocultivation of protoplasts or the transformation of pieces of leaf by agrobacteria is used.

For this purpose, the plant vector construct is transferred by transformation with purified DNA or, mediated by a helper strain such as E. coli SM10 (Simon R. et al., Biotechnology 1, 784-791 (1983)), in Agrobakterium tumefaciens such as A 282 via triparental mating using a Ti plasmid. Direct transformation and triparental mating were carried out as described in, "Plant Molecular Biology Manual" (Kluwer Academic Publishers, Dardrecht (1988)).

Basically all plants can be transformed using the binary plant vectors according to the invention and carrying constructed DNA. Dicotyledonous plants, in particular useful plants, which produce or store for example starch, carbohydrates, proteins or fats in usable amounts in their organs, or produce fruit and vegetables, or provide spices, fibers and technically usable products or medicaments, dyes or waxes, as well as fodder plants are preferred. Examples which may be mentioned are tomato, Sstrawberry, avocado, and plants which carry tropical S 35 fruit, e.g. papaya, mango, but also pear, apple, nectarine, apricot or peach. Furthermore, as examples of 7 plants to be transformed all types of cereal, rape, potatoes, soya bean, cotton, corn, sugar beet or sunflower may be listed.

The transformed cells are selected with the aid of a selection medium, grown to give a callus and regenerated to the plant on an apnropriate medium (Shain et al., Theor. appl. Genet. 72, 770-770 (1986); Masson, J. et al., Plant Science 53, 167-176 (1987); Zhan et al., Plant Mol. Biol. 11, 551-559 (1988); McGranaham et al., Bio/Technology 6, 800-804 (1988); Novrate et al., Bio/Technology 2, 154-159 (1989).

The resulting plant is altered by the transformation insofar as the RNA which is expressed with the aid of the constructed oligonucleotides is cleaved open in the cells at GUC cleavage sites by the ribozyme activity in order to release the genes.

It is also possible to use the described system in bacteria, cell cultures, yeasts or other eukaryotic organisms.

The examples which follow illustrate the invention in more detail.

n

Q^J

8- Examples 1) DNA structures used a) Acetyltransferase gene having Sail linkers 9 OTC GAC ATG TCT CCf3 3, G TAC AGA GGC 54 ACA BCA GCT OAT ATG TGT CST CGA CTA TAC 99 ATT GAG ACG TCT ACA TAA CTC TGC AGA TGT 144 CAA GAG TOO ATT OAT GTT CTC ACC TAA CTA 109 TOG TTG OTT GCT GAO ACC AAC CAA CGA CTC 23.4 OCT 006 CCC TOG AAG, COA CCC 000 ACC, TTC 2479 AST ACT OTT TAC OTG TCA TGA CAA ATO CAC 3,24 TCC ACA TTG TAC ACA AO TOT AAC ATO TOT :569 TTT AAO TCT OTG OTT AAA TTC AGA CAC, CAA 414 OTT AGO TTG CAT GAG CAA TCC AAC GTA CTC 459 COO OCA OCT OGA TAC GO COT CGA CCT ATG 50)4 TOG CAA AGO OAT TTT ACC OTT TCC CTA AAA 549? CCA OTT ACC CPAO ATC GOT CAA TOO OTC TAG 18 GAG AGO AOA CTC TCO TCT 637 0CC 000 OTT COG COO CAA 108 OTO AAC TTT CAC TTO AAA 153 OAT OTA GAG CTA OAT CTO 198 OTT GAO GOT CAA CTO CCA 2473 OCT AGO AAC 004 TOO TTG 288 TCA CAT AGO AST OTA TOO CAT TTO CTT OTA AAC GAA 378 OCT OTT ATA OGA CAA TAT 4 23 OCT TTO OGA CGA AAC COT 468 AAO CAT GOT TTC OTA CCA 5137 27

CA

GOT

72

ACA

117

AGO

TOO

162

AGO

TCC

20)7

OTT

CAA

2 52-

OCT

COA

297

CAT

OTA

342

AAG

TTC

7,e7

GGC

COO

TAC

ATG

477

GSA

OCT

3 64a OTT GAO ATT AGO OCA OCT CAA CTC TAA TCC GOT OGA 81 OAT ATC OTT AAC CAT TAO OTA TAO CAA TTG OTA ATO 126 175 ACA GAG OCA CAA ACA OCA TOT CTC GOT OTT TOT GOT 171 18B0: TTG CAA OAT AGA TAC CCT AAO OTT CTA TCT ATG OGA 216 225 OTO OCT GOT ATT OCT TAO CAC CGA CCA TAA OGA ATO 261 270 TAC OAT TOG ACA OTT GAG ATO CTA ACC TOT CAA CTC 306 3.15 CAA AGO TTG 000 OTA GOA OTT TCC AAC CCOGOAT OCT 3751 360 TOT ATO GAO 000 CAA GOT AGA TAC CTC COO OTT OCA 396 405 CTT OCA AAC OAT COA TCT OAA GOT TTO CTA GOT AGA 441 4 ACA 0CC CGS GOT ACA TTO TOT COG 0CC CCA TOT AAC 486 495 TOO CAT OAT OTT GOT TTT ACC OTA OTA CAA OCA AAA 57.1 540 OCT CCA AGO OCA OTT AGO GGA GOT TCC GGr CAA TOO GAO TTG OCA OCT CTC AAC GOT CGA 558 TGA G- 3 ACT CAG CTG b) Spacer having Sail and HindIII linkers 9 18 27 Z6 TOG ACT TIAC 000 TAA AAT GOT CAG TAT CCC, OCA AAG 000 0CC SC0Z' 71' GA ATO CO ATT TTA OCA OTC ATA 000 GOT TTO COO COO CST trX 9 c) Cat gene from Tn9 according to Alton et al.

Nature 282, 864-866 (1979) d) Ribozyme structure dcmain having HindIII and PstI linkers 9 18 27 36 AGC TGC GGC CGC TTA CGG CTA AAA TGG TCA GTA TCC CCC AAA GGG 3' CG CCG GCG AAT GCC GAT TTT ACC AGT CAT AGG GGG .TTT CCC 54 63 72 81 GTA CCC CTT TCG GGC ATA CTC TGA TGA GTC CGT GAG GAC GAA ACC CAT GGG GAA AGC CCG TAT GAG ACT ACT CAG GCA CTC CTG CTT TGG 99 108 ATT TTA GCC GTA ACT GCA 3' TAA AAT CGG CAT TG The oligonucleotides under b) and d) were synthesized by means of a DNA synthesizer by the phosphoramidite method.

2) Cloning the fragments The DNA specified under la)-d) was ligated in equal molar amounts and incorporated into the SalI/PstI sites of the vector pDH51. Positive clones were identified by hybridization with all 4 radioactively labeled DNA sections used.

3) Cloning into pOCA18 T'e plasmid pOCA18 is reproducibly described in Olszewski, N. et al. NAR 16, 10765-10782 (1988).

An NosI/HindIII fragment with a length of 2.4 kbp was isolated from the described vector pDH51 with the inserted construction and, after filling in the ends, cloned into a pOCA18 vector which had been cut using Bam HI and filled in. Positive clones were detected by hybridization with 32 P-labeled DNA.

10 4) Transformation of agrobacteria The pOCA18 vector with the described 35S promoter/insert was transferred into the agrobacteria strain A 282 (Pharmacia Freiburg, FR Germany, or ATCC 37349, USA).

This was carried out by triparental mating with the aid of the E. coli strain SM10 (Simon, R. et al.

Bio/Technology 1, 784-791, 1983). For this purpose, equal amounts of the bacteria were applied together onto a filter overnight, rinsing with 2 ml of 10 mM MgSO 4 was carried out and aliquots thereof were applied to YEB plates containing tetracycline and rifampicin (YEB: 1 yeast extract, 1 peptone, 0.5 NaC1). It was possible to detect positive agrobacteria by hybridization.

Transformation of tobacco The agrobacteria were grown in YEB medium (1 yeast extract, 1 peptone, 0.5 NaCl) containing tetracycline and rifampicin. 20 ml of the bacteria were spun down, washed once in YEB medium and suspended in 20 ml of 10 mM MgSO 4 in a PetiL dish. The plant material used was Nicotiana tabacum Wisconsin 38. The plants had been cultivated for 4 weeks under sterile conditions on 2MS medium Murashige T. et al., Physiol.. Plant 15, 473-497 (1962) at 25 0 C with 16 hours of light per day. A 1 cm 2 leaf piece was cut off from these plants, wounded using sterile emery paper and immersed in the bacteria culture for 30 sec. The leaf pieces were maintained on MS medium, as described above for 2MS, at 25°C for 2 days and were then washed with liquid 2MS medium. The leaf pieces were then transferred onto MSC 10 plates (as MS containing 1.5 agar) containing 100 pg/ml of kanamycin. After 56 weeks, it was possible to replant regenerate' plants into larger vessels where they formed roots after S 23 weeks.

41 1,i~h. 11 6) Detection of transformation DNA was isolated from transformed tobacco plants with an age of about 8 weeks using standard methods (Maniatis et al., Lab. Journal), transferred to nitrocellulose membranes and hybridized with 32 P-labeled insert DNA.

It was possible to demonstrate an incorporation of the desired sequences in the DNA of the plant.

7) Detection of the expression of the RNA RNA was isolated from the abovementioned tobacco plants from a second leaf sample, was transferred from a formaldehyde gel to nitrocellulose and hybridized as above. It was possible to detect several bands which showed the expected sizes.

8) Detection of in vitro function of the ribozyme RNA The multifunctional RNA was produced from the pBluescript SK+ clones containing the inserted entire oligo using T3 or T7 polymerase in a reaction mixture (Stratagene, Product Information for SK+) and was then isolated. Hybridization of this RNA with individual components showed that the RNA was cleaved open.

9) Detection of in vivo activity of the genes Transformed plants showed growth on 2MS medium containing phosphinotricine. In a spray experiment, the plants likewise proved to be resist. An experiment in order to acetylate chloramphenicol showed that the plants express an active enzyme.

e,

Claims

1. A hybrid gene comprising one or mr copies of a gene sequence coding for ribozyme RNA and of one or various functional genes, the genes sequences being linked in each case via a spacer which, on the RNA level, represents a substrate for the ribozyme.

2. A hybrid gene as claimed in claim 1, wherein the func- tional gene codes for phosphinothricin acetyltransferase and/or for chloramphenicol acetyltransferase.

3. A host cell containing a gene as claimed in claim 1 or 2.

4. A microorganism or a plant, plant cell, and a part or a seed of the plant, containing a gene as claimed in claim 1 or 2. /!i